Role of Endocytic Inhibitory Drugs on Internalization of Amyloidogenic Light Chains by Cardiac Fibroblasts
2006; Elsevier BV; Volume: 169; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.060183
ISSN1525-2191
AutoresGrace Fortes Monis, Christopher Schultz, Ruiyi Ren, Jeremy Eberhard, Catherine E. Costello, Lawreen H. Connors, Martha Skinner, Vickery Trinkaus‐Randall,
Tópico(s)Skin and Cellular Biology Research
ResumoAmyloidosis is a disease of protein misfolding that ultimately impairs organ function. Previously, we demonstrated that amyloidogenic light chains (κ1, λ6, and λ3 subtypes), internalized by cardiac fibroblasts, enhanced sulfation of secreted glycosaminoglycans. In this study, we investigated the inter-nalization and cellular trafficking of urinary immunoglobulin light chains into cardiac fibroblasts. We demonstrate that these light chains have the ability to form annular rings in solution. Internalization was assessed by incubating cells in the presence of light chain conjugated to Oregon Green 488 followed by monitoring with live cell confocal imaging. The rate of light chain internalization was reduced by treatment with methyl-β-cyclodextrin but not filipin. Amyloid light chain did co-localize with dextran-Texas Red. Once internalized, the light chains were detected in lysosomes and then secreted into the extracellular medium. The light chain detected in the cell lysate and medium possessed a lower hydrophobic species. Nocodazole, a microtubule inhibitor, did not disperse aggregates. In addition, internalization and retention of the light chain proteins was altered in the presence of the proteasomal inhibitor MG132. These results indicate that the cell internalizes light chain by a fluid phase endocytosis, which is then modified and ultimately compro-mises the cell. Amyloidosis is a disease of protein misfolding that ultimately impairs organ function. Previously, we demonstrated that amyloidogenic light chains (κ1, λ6, and λ3 subtypes), internalized by cardiac fibroblasts, enhanced sulfation of secreted glycosaminoglycans. In this study, we investigated the inter-nalization and cellular trafficking of urinary immunoglobulin light chains into cardiac fibroblasts. We demonstrate that these light chains have the ability to form annular rings in solution. Internalization was assessed by incubating cells in the presence of light chain conjugated to Oregon Green 488 followed by monitoring with live cell confocal imaging. The rate of light chain internalization was reduced by treatment with methyl-β-cyclodextrin but not filipin. Amyloid light chain did co-localize with dextran-Texas Red. Once internalized, the light chains were detected in lysosomes and then secreted into the extracellular medium. The light chain detected in the cell lysate and medium possessed a lower hydrophobic species. Nocodazole, a microtubule inhibitor, did not disperse aggregates. In addition, internalization and retention of the light chain proteins was altered in the presence of the proteasomal inhibitor MG132. These results indicate that the cell internalizes light chain by a fluid phase endocytosis, which is then modified and ultimately compro-mises the cell. The dynamics of cellular processing of light chains (LCs) has been of great interest to investigators, but the cellular response is not well understood. Primary (AL) amyloidosis is a disease of protein misfolding. Plasma cell dyscrasia produces amyloidogenic immunoglobulin LCs that circulate through the vascular system and deposit as insoluble fibrils in tissues. The nonbranching fibrils are composed of filaments that form β-pleated sheets, stain with Congo Red, and display an apple-green birefringence with polarizing microscopy. The LCs are known to disrupt the normal physiology of organs such as heart, kidney, lungs, peripheral nerves, and intestines, with the most common involvement in kidney and heart. Although renal amyloidosis often results in nephrotic syndrome, cardiac involvement occurs in up to 50% of patients with AL amyloidosis and is a leading cause of morbidity.1Pascali E Diagnosis and treatment of primary amyloidosis.Crit Rev Oncol Hematol. 1995; 19: 149-181Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 2Falk RH Comenzo RL Skinner M The systemic amyloidoses.N Engl J Med. 1997; 337: 898-909Crossref PubMed Scopus (1069) Google Scholar Congestive heart failure is associated with amyloidotic cardiomyopathy.2Falk RH Comenzo RL Skinner M The systemic amyloidoses.N Engl J Med. 1997; 337: 898-909Crossref PubMed Scopus (1069) Google Scholar Investigators have demonstrated that LCs induce oxidative stress responses in isolated cardiac cells and are associated with a decrease in contractility.3Brenner DA Jain M Pimentel DR Wang B Connors LH Skinner M Apstein CS Liao R Human amyloidogenic light chains directly impair cardiomyocyte function through an increase in cellular oxidant stress.Circ Res. 2004; 94: 1008-1010Crossref PubMed Scopus (294) Google Scholar These data indicate that LC toxicity may contribute to cardiac dysfunction as well as play a role in amyloid fibril deposition. Furthermore, internalization of LCs (κ1, λ6, and λ3 subtypes) by cardiac fibroblasts resulted in enhanced sulfation of secreted glycosaminoglycans (GAGs) with minimal heparan-sulfated proteoglycans localized within the cytoplasm.4Trinkaus-Randall V Walsh MT Steeves S Monis G Connors LH Skinner M Cellular response of cardiac fibroblasts to amyloidogenic light chains.Am J Pathol. 2005; 166: 197-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar These results supported earlier studies demonstrating an increase in GAGs with amyloid deposition.5Ancsin JB Amyloidogenesis: historical and modern observations point to heparan sulfate proteoglycans as a major culprit.Amyloid. 2003; 10: 67-79Crossref PubMed Scopus (133) Google Scholar However, the mechanism of internalization and cellular trafficking of LCs is not well understood. Internalization and cellular trafficking has been studied using a number of amyloid systems. 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Our goal was to evaluate the internalization and processing of soluble LCs by cardiac fibroblasts. Cardiac fibroblasts were chosen because of their role in the enhanced production of glycosaminoglycans in response to LCs, and the known association of GAGs and amyloid deposits.4Trinkaus-Randall V Walsh MT Steeves S Monis G Connors LH Skinner M Cellular response of cardiac fibroblasts to amyloidogenic light chains.Am J Pathol. 2005; 166: 197-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Yang AJ Chandswangbhuvana D Margol L Glabe CG Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Abeta1-42 pathogenesis.J Neurosci Res. 1998; 52: 691-698Crossref PubMed Scopus (242) Google Scholar, 24Long CS Henrich CJ Simpson PC A growth factor for cardiac myocytes is produced by cardiac nonmyocytes.Cell Regul. 1991; 2: 1081-1095Crossref PubMed Scopus (144) Google Scholar Rat primary cardiac fibroblasts were treated with fluorescently labeled LCs in the presence of endocytic inhibitors, proteasomal inhibitors, and cellular probes. The response of the cells and the localization of the LCs were evaluated. We established that the rate of internalization was concentration and mass-dependent and was reduced by treatment with methyl-β-cyclodextrin (MβCD) but not by filipin. Co-localization is detected with dextran-Texas Red, a marker of pinocytosis. In addition, proteasomal inhibitors altered processing of the LC, resulting in increased retention.25Goldsmith EC Hoffman A Morales MO Potts JD Price RL McFadden A Rice M Borg TK Organization of fibroblasts in the heart.Dev Dyn. 2004; 230: 787-794Crossref PubMed Scopus (244) Google Scholar, 26Voorhees PM Orlowski RZ The proteasome and proteasome inhibitors in cancer therapy.Annu Rev Pharmacol Toxicol. 2006; 46: 189-213Crossref PubMed Scopus (219) Google Scholar LCs were purified from urine collected from patients with AL amyloidosis with the approval of the Institutional Review Board of Boston University Medical Center. A total of four κ1 and one λ6 LCs were evaluated. The κ LCs included two monomers, a truncated form, and a dimer. The λ6 LC was a monomer. All LCs examined here had been evaluated previously and had demonstrated internalization.4Trinkaus-Randall V Walsh MT Steeves S Monis G Connors LH Skinner M Cellular response of cardiac fibroblasts to amyloidogenic light chains.Am J Pathol. 2005; 166: 197-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Urine samples were dialyzed, lyophilized, and treated with Affi-Gel Blue (Bio-Rad Laboratories, Hercules, CA) to remove albumin. LC proteins were purified by chromatographic separation on a Sephacryl S-200 column (Amersham Pharmacia Bio-Tech, Buckingham, UK). The immunoglobulin LC proteins were subtyped using a number of cellular and molecular analyses and the sequence examined using both molecular and mass spectrometry.27Orlowski RZ Voorhees PM Garcia RA Hall MD Kudrik FJ Allred T Johri AR Jones PE Ivanova A Van Deventer HW Gabriel DA Shea TC Mitchell BS Adams J Esseltine DL Trehu EG Green M Lehman MJ Natoli S Collins JM Lindley CM Dees EC Phase 1 trial of the proteasome inhibitor bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies.Blood. 2005; 105: 3058-3065Crossref PubMed Scopus (283) Google Scholar Primary rat cardiac fibroblasts were isolated as previously described.4Trinkaus-Randall V Walsh MT Steeves S Monis G Connors LH Skinner M Cellular response of cardiac fibroblasts to amyloidogenic light chains.Am J Pathol. 2005; 166: 197-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar In brief, ventricles were subjected to digestion in a buffer containing collagenase, hyaluronidase, and trypsin. Cells were cultured in Dulbecco's low-glucose modified Eagle's medium supplemented with 7% calf serum, 1% nonessential amino acids, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Grand Island, NY).4Trinkaus-Randall V Walsh MT Steeves S Monis G Connors LH Skinner M Cellular response of cardiac fibroblasts to amyloidogenic light chains.Am J Pathol. 2005; 166: 197-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Cells were used in either the first or second passage. For live cell imaging experiments cells were plated at a concentration of 5 × 103 cells/ml on eight-well coverslip chamber slides (Nalge Nunc, Rochester, NY) and cultured for 48 hours. For other assays, cells were plated on P-100s at an equivalent cell density. Imaging was performed on a Zeiss LSM 510 confocal microscope (Thornwood, NY) as described previously.4Trinkaus-Randall V Walsh MT Steeves S Monis G Connors LH Skinner M Cellular response of cardiac fibroblasts to amyloidogenic light chains.Am J Pathol. 2005; 166: 197-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Lim A Wally J Walsh MT Skinner M Costello CE Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry.Anal Biochem. 2001; 295: 45-56Crossref PubMed Scopus (37) Google Scholar, 29Klepeis VE Weinger I Kaczmarek E Trinkaus-Randall V P2Y receptors play a critical role in epithelial cell communication and migration.J Cell Biochem. 2004; 93: 1115-1133Crossref PubMed Scopus (109) Google Scholar Images were taken using a ×63 objective with an optical slice of 3 μm. Live cell imaging was performed with a ×40 objective with an optical slice ranging from 1 to 3 μm. Z-stacks were taken with an optical slice of 1 μm at an interval of 0.5 μm. Images were collected with ×4 averaging. Detector gain and amplitude offset were determined for each experiment to maximize the linear range without saturation and were kept consistent for comparable experiments. Average fluorescence intensities of internalized LCs were measured in individual cells using Zeiss LSM software for region of interest and graphed throughout time. Live cell imaging was performed to monitor the localization and internalization rate of LC proteins. Amyloidogenic LCs were conjugated to Oregon Green 488 (Molecular Probes, Eugene, OR) and purified over a sizing column using the suggested methodology from Molecular Probes and Trinkaus-Randall and colleagues.4Trinkaus-Randall V Walsh MT Steeves S Monis G Connors LH Skinner M Cellular response of cardiac fibroblasts to amyloidogenic light chains.Am J Pathol. 2005; 166: 197-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The ratio of Oregon Green molecules/LC was determined for each conjugation and used to calculate comparative internalization rates between different LCs. OG488:LC = A496 · 100/70,000 · Cp, where Cp is LC molar concentration. The fluorescently conjugated LCs (1.29 μmol/L) were added to cells plated on eight-well glass coverslips and imaged at an excitation of 488 nm for interval time points throughout a period of 24 hours. A single Z-series was taken at each experiment to determine the localization of the LC within the cell compared with a simultaneously acquired differential interference contrast image. The addition of unconjugated Oregon Green 488 to cells was used as a control. Indirect immunohistochemistry was performed to localize specific proteins after fixation.30Richardson TP Trinkaus-Randall V Nugent MA Regulation of heparan sulfate proteoglycan nuclear localization by fibronectin.J Cell Sci. 2001; 114: 1613-1623Crossref PubMed Google Scholar, 31Song QH Gong H Trinkaus-Randall V Role of epidermal growth factor and epidermal growth factor receptor on hemidesmosome complex formation and integrin subunit beta4.Cell Tissue Res. 2003; 312: 203-220Crossref PubMed Scopus (20) Google Scholar, 32Trinkaus-Randall V Tong M Thomas P Cornell-Bell A Confocal imaging of the alpha 6 and beta 4 integrin subunits in the human cornea with aging.Invest Ophthalmol Vis Sci. 1993; 34: 3103-3109PubMed Google Scholar In brief, cells were fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.2 for 15 minutes at room temperature, washed with PBS, permeabilized with 0.1% Triton X-100, and blocked with PBS containing 5% bovine serum albumin. The cells were incubated in PBS/1% bovine serum albumin containing appropriate monoclonal antibodies (mAbs) for 18 hours at 4°C. After incubation with the primary antibody, cells were rinsed with PBS, washed in 3% bovine serum albumin/PBS, and incubated with a secondary antibody conjugated to Alexa Fluor 546 or 633 (1:250) (Molecular Probes) for 2 hours at room temperature or co-stained with rhodamine phalloidin (1:50) for 45 minutes at room temperature. Confocal settings were optimized to control for signal crossover. Cells were imaged using the LSM 510 version 3.2 as described.32Trinkaus-Randall V Tong M Thomas P Cornell-Bell A Confocal imaging of the alpha 6 and beta 4 integrin subunits in the human cornea with aging.Invest Ophthalmol Vis Sci. 1993; 34: 3103-3109PubMed Google Scholar, 33Trinkaus-Randall V Kewalramani R Payne J Cornell-Bell A Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi.J Cell Physiol. 2000; 184: 385-399Crossref PubMed Scopus (19) Google Scholar The internalization of LCs by cardiac fibroblasts was evaluated in the presence of inhibitors of endocytosis. The following optimal conditions were established to maintain morphological integrity and demonstrate efficacy of the drug: 10 mmol/L methyl-β-cyclodextrin (MβCD), 1 hour pretreatment; 10 μmol/L cytochalasin D, 30 minutes pretreatment; 1 μmol/L nocodazole, 1 hour pretreatment; 7.6 μmol/L filipin, 1 hour pretreatment; and 5 μmol/L MG132 for 1 hour pretreatment (Sigma, St. Louis, MO). Light Chain-OG was added to cells in the presence or absence of inhibitors and followed throughout time. To monitor intracellular trafficking of LCs, fluorescent markers of membrane structures and mechanisms were used. The following fluorescent live cell markers were used in conjunction with LC-OG (488): cholera toxin B (555) 0.877 μmol/L for 2 hours, wheat germ agglutinin (WGA) (633) 0.105 μmol/L at several time points at 37°C, Lysotracker DND 99 (577) 50 nmol/L for 30 minutes, Mitotracker 25 nmol/L for 30 minutes, ER Tracker (587) 1 μmol/L for 20 minutes, transferrin 0.312 μmol/L with an 8-minute pulse,34Ghosh RN Gelman DL Maxfield FR Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy.J Cell Sci. 1994; 107: 2177-2189Crossref PubMed Google Scholar FLAER (633) 10−8 mol/L, and FM4-64 8.23 μmol/L for 8 minutes at 4°C at 37°C (Molecular Probes). Dextran-Texas Red (10 and 40 kd) (Molecular Probes) were added to cells at equimolar concentrations as AL-LC for parallel time course experiments. Co-localization was defined using the Zeiss LSM 510 co-localization co-efficient program, images were scanned at the optimal pixel number (2048 × 2048) and the crosshair function and scatter region of interest mode were used. Only pixels above background fluorescence were evaluated using LSM 510 co-localization and crosshair functions.35Manders EM Verbeek FJ Aten JA Measurement of co-localization of objects in dual-colour images.J Microsc. 1993; 169: 375-382Crossref PubMed Scopus (1529) Google Scholar To assess the formation of annular LC complexes, transmission electron microscopy and atomic force microscopy were used. Five μl of 1.1 μmol/L LC solution were applied to 300 mesh carbon-coated copper grids that had been glow discharged rendering the surface hydrophilic. The LC was adsorbed onto the grid for 25 minutes at room temperature, and the excess solution was removed by wicking filter paper. Samples deposited on grids were stained negatively with 1% sodium phosphotungstic acid (NaPT) for 10 seconds. NaPT was removed and the sample air-dried. Grids were visualized with a Philips CM12 transmission electron microscope. Atomic force microscopy imaging was performed with a commercial MultiMode atomic force microscope controlled by Nanoscope IIIa electronics (Digital Instruments, Santa Barbara, CA) equipped with a 12-μm scanner (E-scanner). Images were taken in air using a tapping mode on the mica surface. RTESP tapping mode silicon tips were mounted on triangular 100-μm-long cantilevers (k = 20 to 80 N/m) (Digital Instruments). The tips were exposed to UV light before use. For high-resolution imaging, the microscope head was placed on a vibration-isolated air table. Samples were imaged by adding the same concentration onto freshly cleaved mica. Samples were incubated for 3 minutes at room temperature, washed with dH2O, and dried under a nitrogen stream. The hydrophobic nature of AL-LC-OG in cell culture media and lysate samples was assessed by RP-HPLC. Cell lysates and medium samples were separated on a gel filtration column (GF-250) to assess for change in mass. Analyses were performed on the samples on a Poroshell 300SB-C8 (5 μm, 2.1 × 75 mm) reversed-phase column (Agilent, Palo Alto, CA) using a linear gradient of 0 to 100% solvent B throughout 5 minutes and operating at a flow rate of 1.0 ml/minute (solvent A: 5% CH3CN, 10 mmol/L Tris, pH 7.4; solvent B: 85% CH3CN, 10 mmol/L Tris, pH 7.4). Elution profiles were generated with fluorescence monitoring (excitation wavelength, 488 nm; emission wavelength, 530 nm). Peak fractions were collected and dried in a SPD111V centrifugal concentrator (ThermoSavant, Holbrook, NY). The presence of LC-OG proteins was evaluated by SDS-PAGE and Western blot analysis. Dried fractions collected from RP-HPLC separations were reconstituted in SDS-PAGE reducing buffer (10 mmol/L Tris-HCl, 1 mmol/L ethylenediaminetetraacetic acid, 2.5% SDS, 5% β-mercaptoethanol, and 0.01% bromphenol blue), heat-denatured with boiling for 3 minutes, electrophoresed on 10 to 15% polyacrylamide gradient gels, and stained with Coomassie blue or immunoblotted. Nitrocellulose membranes were probed with rabbit anti-human antibodies directed against κ1-LC (Dako Cytomation, Carpinteria, CA) and then incubated with a goat anti-rabbit whole IgG antibody conjugated to alkaline phosphatase (Sigma). Blots were visualized using BCIP/NBT color development substrate (Promega, Madison, WI). SDS-PAGE and transfer of HPLC samples were performed on the PhastSystem (GE Health Care, Giles, UK). Other lysates were evaluated using SDS-PAGE, and the LC-OG was detected using an antibody directed against Oregon Green (Molecular Probes), or the LC-OG was imaged directly on the KODAK Image Station 440 (Eastman-Kodak, Rochester, NY). Formation of pore-like structures have been demonstrated with a number of proteins associated with protein misfolding. Our goal was to determine whether annular rings formed from κ1-LCs in solution in our system. The change from oligomers to ring-like structures was detected using atomic force microscopy and transmission electron microscopy (Figure 1). The atomic force microscopy image represents an annular ring formed in solution (0.25 mg κ1-LC, pH 2, 25°C). It has an average outer/inner diameter of 68.01 nm/30.07 nm, respectively (Figure 1). At this level of resolution (500 nm × 500 nm), three subunits were detected. Rings were detected throughout a range of pH (2 to 7) and were a minor species (1%). To image larger surface areas, negative staining transmission electron microscopy was used. The experiments were performed under conditions determined to be optimal for fibril formation (without the addition of other components such as GAGs) (0.25 mg/ml κ1-LC, pH 6, 37°C, rotation of 25 rpm). Two types of ring-like structures were observed; one with a larger ring (average outer/inner diameter, 110.5/42.5 nm) and a second smaller ring with a dimension similar to the atomic force microscopy (average outer/inner diameter, 76.5/36 nm). These data indicate that soluble LCs purified from clinical sources (see Materials and Methods) are capable of forming annular ring structures in vitro. To determine internalization rate, fluorescence intensity of internalization of κ1-LC-OG was measured as described in the Materials and Methods, reflecting the number of Oregon Green molecules bound per LC. Images taken every minute throughout a period of 1 hour demonstrated a gradual increase in internalized LC-OG signal, shown in representative images (Figure 2A). The κ1-LC displayed a cytoplasmic, punctate pattern with an increase in perinuclear concentration throughout time (Figure 2A). When the images were run continuously as movies, the internalized LCs displayed continual motion (average rate, 0.25 um/second) (Supplemental Movie 1, see http://ajp.amjpathol.org). By 4 hours, the punctae had a mean volume of 0.5 μm3. The motion was arrested when cells were treated with nocodazole, a microtubule inhibitor. In addition, the punctae appeared to form small aggregates in the presence of nocodazole, which had a mean volume of 0.75 μm3. There was no dispersion of the aggregates detected throughout time in the presence of the microtubule inhibitor. The internalization depended on concentration of the LC, mass, and temperature. It correlated positively with increasing concentration of κ1-LC. When cells were exposed to increasing concentrations of protein (0 to 5.15 μmol/L), the internalization rate (change in intracellular fluorescence intensity) did not plateau throughout a period of 4 hours (Figure 3A). Internalization was verified in cell lysates using HPLC, SDS-PAGE, and Western blot analysis using an antibody directed against the Oregon Green of the conjugated LC-OG (Figure 10, Figure 11). After 24 hours, there was a decrease in the rate of internalization, which we hypothesized to be attributable to secretion of the LC. To evaluate secretion, cells were incubated in the presence of fluorescently labeled LC for 24 hours, and the medium was replaced with LC-deficient medium. Both the intracellular fluorescence and the fluorescence in the medium were monitored. As predicted, a decrease in intracellular LC fluorescence was detected after the removal of LC-rich medium (Figure 3B). After 3 days, LC-OG was detected in the medium at a concentration of 19.6 nmol/L.Figure 10Change in hydrophobicity of LC-OG after internalization. Cardiac fibroblasts were grown to confluency in 100-mm2 plates and exposed to LC-OG for 3 days. Samples were separated on a GF-250 and analyses performed on a Poroshell 300SB-C8 RP column using a linear gradient of 0 to 100% acetonitrile. Elution profiles were generated with fluorescence monitoring (excitation, 488; and emission, 530 nm). Early peaks represent void and unconjugated OG. LC-OG cell lysate at day 0 containing internalized LC (—-), pu
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